Determination of the amount of magnesium ions is generally classified into two methods; one is a physicochemical determination, especially an atomic-absorption spectrophotometry method, and the other is a chemical determination such as a method employing xylidyl blue. The physicochemical determination can give accurate results, but it needs an expensive analyzer and the operations of the analyzer are very complicated. The chemical determination can not give accurate results, because the specificity of reagents to be employed is so low that the reagents tend to react with other materials than magnesium ions.
In order to overcome the above mentioned problems, there is proposed a reagent for determining the amount of magnesium ions comprising hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PBH), in Methods of Enzymatic Analysis Third Edition, pages 529 to 597 (1985). The reagent utilizes a reaction system wherein the magnesium ions are essential for obtaining activity in the coupled reaction of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH). The reaction mechanism is as follows: ##STR1##
Wherein ATP represents adenosine-5'-triphosphate, ADP represents adenosine-5'-diphosphate, and NAD(P).sup.+ represents oxidized-form nicotinamide adeninedinucleotide(phosphate). In the reaction mechanism, hexokinase (HK) does not exhibit activity to ATP until the magnesium ions are reacted with adenosine-5'-triphosphate (ATP) and changed to ATP-Mg, because ATP-Mg is a substrate of hexokinase (HK) but adenosine-5'-triphosphate (ATP) is not. Accordingly, in order to determine the amount of the magnesium ions, the concentration of adenosine-5'-triphosphate (ATP) must be adjusted to more than that of the magnesium ions. Otherwise, only a portion of magnesium ions is converted into ATP-Mg and the remaining magnesium ions do not take part in the above reaction mechanism.
On the other hand, hexokinase (HK) also has an adenosinetriphosphatase (ATP ase) activity by which adenosine-5'-triphosphate (ATP) is hydrolyzed into adenosine-5'-diphosphate (ADP) and thus, in the reaction mechanism, the concentration of adenosine-5'-triphosphate (ATP) tends to decrease by an attack of hexokinase (HK). As a result, it becomes difficult to adjust the concentration of adenosine-5'-triphosphate (ATP) to more than that of the magnesium ions. Accordingly, an accurate determination of the amount of magnesium ions is difficult.
Japanese Patent Publication (unexamined) 169598/1981 discloses a measuring composition comprising glucokinase (Glck) and glucose-6-phosphate dehydrogenase (G6PDH). The composition is employed for determining the amount of glucose or creatine kinase, but not for magnesium ions.
It has been found by the present inventors that glucokinase (Glck) indicates a high specificity to glucose but does not indicate an adenosinetriphosphatase (ATP ase) activity to adenosine-5'-triphosphate (ATP), and that, when the glucokinase (Glck) is coupled with glucose-6-phosphate dehydrogenase (G6PDH), magnesium ions can be accurately determined. There are no reports disclosing that the coupled reaction of glucokinase (Glck) and glucose-6-phosphate dehydrogenase (G6PHD) is utilized for determining the amount of magnesium ions.